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Multiple mutations in the Rhodopsin gene cause sector retinitis pigmentosa in humans and a corresponding light-exacerbated retinal degeneration (RD) in animal models. Previously we have shown that T4K rhodopsin requires photoactivation to exert its toxic effect. Here we further investigated the mechanisms involved in rod cell death caused by T4K rhodopsin in mixed male and female Xenopus laevis In this model, RD was prevented by rearing animals in constant darkness but surprisingly also in constant light. RD was maximized by light cycles containing at least 1 h of darkness and 20 min of light exposure, light intensities >750 lux, and by a sudden light onset. Under conditions of frequent light cycling, RD occurred rapidly and synchronously, with massive shedding of ROS fragments into the RPE initiated within hours and subsequent death and phagocytosis of rod cell bodies. RD was minimized by reduced light levels, pretreatment with constant light, and gradual light onset. RD was prevented by genetic ablation of the retinal isomerohydrolase RPE65 and exacerbated by ablation of phototransduction components GNAT1, SAG, and GRK1. Our results indicate that photoactivated T4K rhodopsin is toxic, that cell death requires synchronized photoactivation of T4K rhodopsin, and that toxicity is mitigated by interaction with other rod outer segment proteins regardless of whether they participate in activation or shutoff of phototransduction. In contrast, RD caused by P23H rhodopsin does not require photoactivation of the mutant protein, as it was exacerbated by RPE65 ablation, suggesting that these phenotypically similar disorders may require different treatment strategies.

Asunto(s)

Degeneración Retiniana , Rodopsina , Xenopus laevis , Animales , Rodopsina/metabolismo , Rodopsina/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/genética , Femenino , Masculino , Fototransducción , Luz/efectos adversos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , cis-trans-Isomerasas/metabolismo , cis-trans-Isomerasas/genética , Muerte Celular

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PURPOSE: This study aimed to investigate the protective or therapeutic effect of thymoquinone (TQ) in a retinal degeneration rat model and its relationships with the retina ultrastructure, heme oxygenase 1 (HO-1), caspase-3, and RPE65 expressions and to determine whether TQ has a therapeutic effect at the biochemical level. METHODS: A total of 25 adult Wistar albino rats were divided into the following treatment groups: saline (control: CONT), CO (corn oil), sodium iodate (SI), TQ + SI, and SI + TQ injection groups. Retina morphology, RPE65, HO-1, and caspase-3 expression levels were evaluated using immunohistochemistry, and optical density was determined using ImageJ. Ultrastructural evaluations were performed with electron microscopy. Thiol-disulfide homeostatic parameters were examined in serum samples. RESULTS: Outer nuclear layer (ONL) thickness was significantly higher in the SI + TQ group compared to the SI group. The RPE65 expression significantly decreased in the SI group compared with the CONT and CO groups. A significant increase in RPE65 expression level and a significant decrease in caspase-3 expression level were found in the SI + TQ group compared with the SI group. The increase in HO-1 expression level was significantly higher in the TQ treatment groups, particularly in the SI + TQ group. In the SI and TQ + SI groups, the ONL thickness significantly decreased with a significant increase in caspase-3 expression compared to the CONT and CO groups. In the treatment groups, decreased organelle damage was observed on electron microscopy. In the SI + TQ group, the disulfide/native thiol and disulfide/total thiol ratios were significantly lower than all other groups, while the native/total thiol ratio was significantly higher than the other experimental groups. CONCLUSIONS: The present study provides evidence that continuous TQ treatment can increase HO-1 and RPE65 expression and decrease apoptosis (caspase-3 levels), thereby preserving the retina at the ultrastructural level. Moreover, TQ administration can maintain thiol/disulfide homeostasis in SI-induced retinal degeneration-modelled rats.

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Purpose: To investigate the short-term effects of low-level lasers (LLLs; also known as low-power laser therapy) on the structure, genetic, and phenotype of cultured human retinal pigment epithelial (hRPE) cells from both adult and neonatal sources. Methods: Cultivated adult and neonatal hRPE cells were irradiated with two types of LLL (630 nm and 780 nm), 1 min daily for five consecutive days. Results: An increase in doubling time was observed in 630 nm-irradiated adult hRPE cells (P = 0.032). The gene expression profile revealed increased expression of retinoid isomerohydrolase RPE65 (RPE65) (P < 0.01 for 630 nm laser, P < 0.001 for 780 nm laser) and nestin (NES) (P < 0.01 for 630 nm laser) in neonatal hRPE cells, upregulation of RPE65 (P < 0.001 for 780 nm laser) and paired box 6 (PAX6) (P < 0.001 for 780 nm laser) genes in adult hRPE cells, and reduced expression of actin alpha 2 (ACTA2) in 780 nm-irradiated adult hRPE cells (P < 0.001). Except the significant increase of α -SMA in 780 nm-irradiated neonatal hRPE cells, no significant change was noted in the expressions of other investigated proteins. Conclusion: Short-term irradiation of neonatal and adult hRPE cells with LLLs may induce multipotency at the transcriptional level. Irradiation of neonatal hRPE cells with LLLs can be associated with increased risk of myofibroblastic transformation; however, adult hRPE cells irradiated with the 780 nm laser have minimal risk of myofibroblastic differentiation. It seems that the 780 nm laser may be a promising option for future photobiomodulation in retinal degenerations in adults.

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PURPOSE: To report a case of macular hole and detachment occurring after the subretinal injection of Voretigene Neparvovec (VN) in a patient affected by atypical RPE65 retinal dystrophy with high myopia and its successful surgical management. CASE DESCRIPTION: We report a case of a 70-year-old man treated with VN in both eyes. The best corrected visual acuity (BCVA) was 0.7 LogMar in the right eye (RE) and 0.92 LogMar in the left eye (LE). Axial length was 29.60 mm in the RE and 30.28 mm in the LE. Both eyes were pseudophakic. In both eyes, fundus examination revealed high myopia, posterior staphyloma, and extended retinal atrophy areas at the posterior pole, circumscribing a central island of surviving retina. Both eyes were treated with VN subretinal injection, but a full-thickness macular hole and retinal detachment occurred in the LE three weeks after surgery. The patient underwent 23-gauge vitrectomy with internal limiting membrane (ILM) peeling and the inverted flap technique with sulfur hexafluoride (SF6) 20% tamponade. Postoperative follow-up showed that the macular hole was closed and the BCVA was maintained. CONCLUSIONS: Our experience suggests that patients with atypical RPE65 retinal dystrophy and high myopia undergoing VN subretinal injection require careful management to minimize the risk of macular hole and detachment occurrence and promptly detect and address these potential complications.

Asunto(s)

Terapia Genética , Desprendimiento de Retina , Distrofias Retinianas , Perforaciones de la Retina , cis-trans-Isomerasas , Humanos , Masculino , Perforaciones de la Retina/etiología , Perforaciones de la Retina/genética , Perforaciones de la Retina/cirugía , Anciano , Desprendimiento de Retina/genética , Desprendimiento de Retina/etiología , Desprendimiento de Retina/cirugía , cis-trans-Isomerasas/genética , Distrofias Retinianas/genética , Vitrectomía , Agudeza Visual , Miopía/genética

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In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Asunto(s)

Retina , Epitelio Pigmentado de la Retina , Animales , Perros , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Nestina/metabolismo , Blastocisto/metabolismo , Blastocisto/citología , Biomarcadores/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre/metabolismo , Células Madre/citología , Inmunohistoquímica , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/patología

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The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In this study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibiting comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least twofold greater than those in age-matched rd12 mice. Moreover, FATP4 deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function, and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4 deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.

Asunto(s)

Proteínas de Transporte de Ácidos Grasos , Amaurosis Congénita de Leber , Células Fotorreceptoras Retinianas Conos , Animales , Células Fotorreceptoras Retinianas Conos/metabolismo , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/metabolismo , Amaurosis Congénita de Leber/patología , Ratones , Proteínas de Transporte de Ácidos Grasos/metabolismo , Proteínas de Transporte de Ácidos Grasos/genética , Masculino , Femenino , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismo , cis-trans-Isomerasas/deficiencia , Supervivencia Celular , Ratones Noqueados , Diterpenos , Visión Ocular/fisiología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Retinaldehído

RESUMEN

Vision starts in retinal photoreceptors when specialized proteins (opsins) sense photons via their covalently bonded vitamin A derivative 11cis retinaldehyde (11cis-RAL). The reaction of non-enzymatic aldehydes with amino groups lacks specificity, and the reaction products may trigger cell damage. However, the reduced synthesis of 11cis-RAL results in photoreceptor demise and suggests the need for careful control over 11cis-RAL handling by retinal cells. This perspective focuses on retinoid(s) synthesis, their control in the adult retina, and their role during retina development. It also explores the potential importance of 9cis vitamin A derivatives in regulating retinoid synthesis and their impact on photoreceptor development and survival. Additionally, recent advancements suggesting the pivotal nature of retinoid synthesis regulation for cone cell viability are discussed.

Asunto(s)

Retinoides , Animales , Humanos , Retina/metabolismo , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Retinaldehído/metabolismo , Retinoides/metabolismo , Vitamina A/metabolismo

RESUMEN

INTRODUCTION: Mutations in the retinal pigment epithelial 65 kilodalton protein (RPE65) gene are associated with various inherited retinal diseases (IRDs), including Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), and retinitis pigmentosa (RP). We screened for mutations in RPE65 in a series of Indian patients with these IRDs to determine the frequency/types of mutations and to describe the associated phenotypes. MATERIALS AND METHODS: Diagnosis of LCA, EOSRD, and RP was made by standard and pre-defined criteria. Patients were evaluated by clinical, retinal imaging, and electrophysiological parameters. Genomic DNA from patients and available family members were used for identifying mutations by direct Sanger sequencing of the RPE65 gene or targeted NGS gene panel for IRDs covering 260+ genes. Variations detected were tested in healthy control populations and for co-segregation with the disease in available family members. RESULTS: Mutations were found in eight patients, out of 220 total cases screened, all homozygous for the respective mutant alleles. Seven patients had mutations leading to premature termination codons and one patient had a missense change. The onset of visual loss ranged from birth to <2 years of life. At presentation, RPE mottling in the background retina was present in all cases with macular involvement in five cases with or without vascular attenuation and optic disc pallor. CONCLUSION: RPE65 mutations in this series were found in 3.6% of cases associated with severe, early-onset disease, with consistent RPE mottling and variable manifestations with regard to the extent of disc pallor, arteriolar attenuation, and appearance of the macula.

Asunto(s)

Amaurosis Congénita de Leber , Mutación , Distrofias Retinianas , Retinitis Pigmentosa , cis-trans-Isomerasas , Humanos , cis-trans-Isomerasas/genética , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/diagnóstico , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/diagnóstico , Masculino , India/epidemiología , Femenino , Distrofias Retinianas/genética , Distrofias Retinianas/diagnóstico , Adulto , Niño , Centros de Atención Terciaria , Preescolar , Linaje , Adolescente , Electrorretinografía , Adulto Joven , Agudeza Visual/fisiología , Análisis Mutacional de ADN , Lactante , Enfermedades Hereditarias del Ojo

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Purpose: To report the effect of internal limiting membrane (ILM) peeling prior to Voretigen Neparvovec-ryzl (VN) subretinal injection on focal chorioretinal atrophy development in patients presenting with RPE65-mediated Leber congenital amaurosis (LCA). Design: Retrospective case series. Methods: Three patients who underwent bilateral subretinal VN injection for RPE65-mediated LCA were followed up for 18-24 months. ILM peeling was performed unilaterally in patients 1 and 2 and bilaterally in patient 3. Chorioretinal atrophy was identified on fundus biomicroscopy, non-mydriatic retinography and/or ultrawide field fundus imaging. Best corrected visual acuity (BCVA), spectral-domain optical coherence tomography (SD-OCT), visual fields, full-field stimulus threshold (FST) and visual functioning questionnaire score (NEI-VFQ-25) were reported. Outcome measures were changes in BCVA, visual fields, FST, NEI-VFQ-25, and chorioretinal atrophy location. Results: Chorioretinal atrophy at the injection site exclusively developed in eyes which did not undergo prior ILM peeling. In patient 3, bilateral pre-operative nummular chorioretinal alterations progressed toward epithelial atrophic patches in the mid and extreme retinal periphery 18 months after VN injection. BCVA and visual fields improved bilaterally. NEI_VFQ 25 remained stable in patient 1 and improved in patient 2 and 3. FST test improved bilaterally in patient 3. Conclusions: ILM peeling prior to VN injection seems to be a smoother and safer technique to administer VN treatment and may prevent secondary focal atrophy development at the injection site. However, another type of more extended chorioretinal atrophy might exist and could be related to LCA evolution or to incompletely understood adverse effect of VN product.

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Voretigene neparvovec (VN) is the first available gene therapy for patients with biallelic RPE65-mediated inherited retinal dystrophy who have sufficient viable retinal cells. PERCEIVE is an ongoing, post-authorization, prospective, multicenter, registry-based observational study and is the largest study assessing the real-world, long-term safety and effectiveness of VN. Here, we present the outcomes of 103 patients treated with VN according to local prescribing information. The mean (SD) age was 19.5 (10.85) years, 52 (50.5%) were female, and the mean (SD) duration of the follow up was 0.8 (0.64) years (maximum: 2.3 years). Thirty-five patients (34%) experienced ocular treatment-emergent adverse events (TEAEs), most frequently related to chorioretinal atrophy (n = 13 [12.6%]). Eighteen patients (17.5%; 24 eyes [13.1%]) experienced ocular TEAEs of special interest, including intraocular inflammation and/or infection related to the procedure (n = 7). The mean (SD) changes from baseline in full-field light-sensitivity threshold testing (white light) at month 1, month 6, year 1, and year 2 were -16.59 (13.48) dB (51 eyes), -18.24 (14.62) dB (42 eyes), -15.84 (14.10) dB (10 eyes), and -13.67 (22.62) dB (13 eyes), respectively. The change in visual acuity from baseline was not clinically significant. Overall, the outcomes of the PERCEIVE study are consistent with the findings of VN pivotal clinical trials.

Asunto(s)

Enfermedades de la Coroides , Retina , Humanos , Femenino , Adulto Joven , Adulto , Masculino , Estudios Prospectivos , Terapia Genética , Sistema de Registros

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PURPOSE: To assess the efficacy of voretigene neparvovec (VN) treatment by objective fixation stability and chromatic pupillometry testing in clinical practice. DESIGN: Retrospective cohort study with longitudinal follow-up. SUBJECTS: Twelve patients (aged 7-34 years) with RPE65-related inherited retinal dystrophies were treated at the same center with VN in both eyes. METHODS: Patients treated at the same center with VN were evaluated over a 12-month posttreatment follow-up by subjective and objective tests. Furthermore, patients treated with VN who developed atrophy were compared with those who did not. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), full-field stimulus threshold test (FST), semiautomated kinetic visual field (SKVF), microperimetry, and chromatic pupillometry over a 12-month follow-up. RESULTS: Significant improvements of BCVA (P < 0.001), SKVF (P < 0.05), and FST (P < 0.001) were already observed 45 days after treatment and were maintained at the 12-month timepoint. Fixation stability, assessed by microperimetry, improved significantly (P < 0.05) after treatment. Chromatic pupillometry showed significant improvements (P < 0.05) at the 6- and 12-month timepoints. The increase in maximum pupillary constriction significantly (P < 0.001) correlated with higher retinal sensitivity in FST. Four patients developed multifocal retinal atrophy in both eyes, detected at the 6-month timepoint, but this atrophy was not generally associated with worse visual function outcomes. CONCLUSIONS: This study explores objective outcomes in order to demonstrate the efficacy of VN treatment in addition to the tests normally performed in clinical practice. Our findings show a significant improvement of retinal function both in subjective assessments, such as BCVA, SKVF, and FST, and in objective measurements of fixation stability and maximum pupillary constriction. Moreover, the significant correlation between maximum pupillary constriction and light sensitivity thresholds corroborates the introduction of chromatic pupillometry as an objective test to better assess treatment outcomes in patients with inherited retinal dystrophies. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references in the Footnotes and Disclosures at the end of this article.

Asunto(s)

Distrofias Retinianas , Agudeza Visual , Campos Visuales , cis-trans-Isomerasas , Humanos , Masculino , Femenino , Adolescente , Estudios Retrospectivos , Niño , Adulto , Agudeza Visual/fisiología , Adulto Joven , Estudios de Seguimiento , Campos Visuales/fisiología , cis-trans-Isomerasas/genética , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/fisiopatología , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Resultado del Tratamiento , Terapia Genética/métodos , Pruebas del Campo Visual

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Inherited retinal diseases (IRD) are a leading cause of blindness in the working age population and in children. The scope of this review is to familiarise clinicians and scientists with the current landscape of molecular genetics, clinical phenotype, retinal imaging and therapeutic prospects/completed trials in IRD. Herein we present in a comprehensive and concise manner: (i) macular dystrophies (Stargardt disease (ABCA4), X-linked retinoschisis (RS1), Best disease (BEST1), PRPH2-associated pattern dystrophy, Sorsby fundus dystrophy (TIMP3), and autosomal dominant drusen (EFEMP1)), (ii) cone and cone-rod dystrophies (GUCA1A, PRPH2, ABCA4, KCNV2 and RPGR), (iii) predominant rod or rod-cone dystrophies (retinitis pigmentosa, enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2)), (iv) Leber congenital amaurosis/early-onset severe retinal dystrophy (GUCY2D, CEP290, CRB1, RDH12, RPE65, TULP1, AIPL1 and NMNAT1), (v) cone dysfunction syndromes (achromatopsia (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), X-linked cone dysfunction with myopia and dichromacy (Bornholm Eye disease; OPN1LW/OPN1MW array), oligocone trichromacy, and blue-cone monochromatism (OPN1LW/OPN1MW array)). Whilst we use the aforementioned classical phenotypic groupings, a key feature of IRD is that it is characterised by tremendous heterogeneity and variable expressivity, with several of the above genes associated with a range of phenotypes.

Asunto(s)

Enfermedades Hereditarias del Ojo , Enfermedades de la Retina , Humanos , Distrofias de Conos y Bastones/genética , Distrofias de Conos y Bastones/fisiopatología , Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/fisiopatología , Genotipo , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Amaurosis Congénita de Leber/fisiopatología , Biología Molecular , Fenotipo , Enfermedades de la Retina/genética , Enfermedades de la Retina/fisiopatología , Enfermedades de la Retina/terapia

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PURPOSE: To investigate the changes in retinal arterial architecture after treatment with voretigene neparvovec in patients with retinal dystrophy caused by bi-allelic mutations in the RPE65 gene. METHODS: Sixteen eyes treated with voretigene neparvovec at the University Eye Clinic in Tuebingen, Germany, underwent adaptive optics ophthalmoscopy (AO) imaging at baseline and 2 weeks, 1, 3, 6 and 12 months after treatment. Follow-up was performed in six eyes of four patients. For each eye, five different positions at arterial vessels were selected and the wall-to-lumen ratio (WLR), the lumen diameter (LD) and the wall cross-sectional area (WCSA) were measured by the manufacturer's software over the observational period. RESULTS: Vast retinal atrophy dominated all gained AO images. WLR fluctuated in the observation period without statistically significant change. LD and WCSA changed significantly after 2 weeks from the baseline examination and returned to values similar to baseline thereafter. There were no signs of inflammation such as macrophages or perivascular accumulated fluid visible. CONCLUSION: AO imaging of the retinal vessels in RPE65-associated retinal dystrophies (IRD) is challenging. There was no change in the retinal arterial vasculature over the observation period of 12 months that would indicate inflammatory changes. Decrease of the LD and WCSA shortly after treatment might be caused by the perioperative prednisolone intake. AO of retinal vessels can be used as a diagnostic module to complement monitoring the disease and effects of genetic treatments if the acquisition is possible in selected cases.

Asunto(s)

Arteriolas , Distrofias Retinianas , Vasos Retinianos , Humanos , Oftalmoscopía , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética

RESUMEN

PURPOSE: To assess the impact of baseline data on psychophysical and morphological outcomes of subretinal voretigene neparvovec (VN) (Luxturna, Spark Therapeutics, Inc.) treatment. DESIGN: Single-center, retrospective, longitudinal, consecutive case series. PARTICIPANTS: Patients with RPE65-biallelic mutation-associated inherited retinal degeneration (RPE65-IRD) treated between February 2020 and March 2022 with VN and oral immunosuppression according to the manufacturer's recommendation by one surgeon (F.G.H.). METHODS: Retrospective analysis of surgical and clinical records, ancillary testing, and retinal imaging after VN therapy for RPE65-IRD. Descriptive statistics compared data at baseline up to 32 months post-treatment. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), low-luminance VA (LLVA), Goldmann visual fields (GVFs), chromatic full-field stimulus threshold (FST) testing (FST), scotopic and photopic 2-color threshold perimetry (2CTP), and multimodal retinal imaging. RESULTS: Thirty eyes of 19 patients were analyzed (10 pediatric patients < 20 years; 20 adult patients > 20 years of age; overall range: 8-40 years) with a median follow-up of 15 months (range, 1-32). The fovea was completely or partially detached in 16 eyes, attached in 12 eyes, and not assessable in 2 eyes on intraoperative imaging. Median BCVA at baseline was better in the pediatric group (P < 0.05) and did not change significantly independent of age. Meaningful loss of BCVA (≥ 0.3 logarithm of the minimal angle of resolution [logMAR]) occurred in 5 of 18 adult eyes, and a meaningful gain (≥-0.3 logMAR) occurred in 2 of 18 adult and 2 of 8 pediatric eyes. The LLVA and scotopic 2CTP improved considerably in pediatric patients. Scotopic blue FST improved at all ages but more in pediatric patients (8/8 eyes gained ≥ 10 decibels [dB]; P < 0.05). In pediatric patients, median GVF improved by 20% for target V4e and by 50% for target III4e (target I4e not detected). Novel atrophy developed in 13 of 26 eyes at the site of the bleb or peripheral of vascular arcades. Improvements in FST did not correlate with development of chorioretinal atrophy at 12 months. Mean central retinal thickness was 165.87 µm (± 26.26) at baseline (30 eyes) and 157.69 µm (± 30.3) at 12 months (26 eyes). Eight adult patients were treated unilaterally. The untreated eyes did not show meaningful changes during follow-up. CONCLUSIONS: These data in a clinical setting show the effectiveness of VN therapy with stable median BCVA and mean retinal thickness and improvements of LLVA, FST, and 2CTP up to 32 months. Treatment effects were superior in the pediatric group. We observed new chorioretinal atrophy in 50% of the treated eyes. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Asunto(s)

Retina , Distrofias Retinianas , Adulto , Humanos , Niño , Estudios Retrospectivos , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Mutación , Atrofia

RESUMEN

BACKGROUND: Retinal pigment epithelium (RPE) 65 is a key enzyme in the visual cycle involved in the regeneration of 11-cis-retinal. Mutations in the human RPE65 gene cause Leber's congenital amaurosis (LCA), a severe form of an inherited retinal disorder. Animal models carrying Rpe65 mutations develop early-onset retinal degeneration. In particular, the cones degenerate faster than the rods. To date, gene therapy has been used successfully to treat RPE65-associated retinal disorders. However, gene therapy does not completely prevent progressive retinal degeneration in patients, possibly due to the vulnerability of cones in these patients. In the present study, we tested whether leukemia inhibitory factor (LIF), a trophic factor, protects cones in rd12 mice harboring a nonsense mutation in Rpe65. METHODS: LIF was administrated to rd12 mice by intravitreal microinjection. Apoptosis of retinal cells was analyzed by TUNEL assay. The degeneration of cone cells was evaluated by immunostaining of retinal sections and retinal flat-mounts. Signaling proteins regulated by LIF in the retinal and cultured cells were determined by immunoblotting. RESULTS: Intravitreal administration of LIF activated the STAT3 signaling pathway, thereby inhibiting photoreceptor apoptosis and preserving cones in rd12 mice. Niclosamide (NCL), an inhibitor of STAT3 signaling, effectively blocked STAT3 signaling and autophagy in cultured 661W cells treated with LIF. Co-administration of LIF with NCL to rd12 mice abolished the protective effect of LIF, suggesting that STAT3 signaling and autophagy mediate the protection. CONCLUSION: LIF is a potent factor that protects cones in rd12 mice. This finding implies that LIF can be used in combination with gene therapy to achieve better therapeutic outcomes for patients with RPE65-associated LCA.

RESUMEN

Generating animal models can explore the role of new candidate genes in causing diseases and the pathogenicity of a specific mutation in the underlying genes. These animals can be used to identify new pharmaceutical or genetic therapeutic methods. In the present experiment, we developed a rpe65a knock out (KO) zebrafish as a retinitis pigmentosa (RP) disease model. Using the CRISPR/Cas9 system, the rpe65a gene was KO in zebrafish. Two specific single-guide RNAs (sgRNAs) were designed for the zebrafish rpe65a gene. SgRNAs were cloned into the DR274 plasmid and synthesized using in vitro transcription method. The efficiency of Ribonucleoprotein (synthesized sgRNA and recombinant Cas9) was evaluated by in vitro digestion experiment. Ribonucleoprotein complexes were microinjected into one to four-celled eggs of the TU zebrafish strain. The effectiveness of sgRNAs in KO the target gene was determined using the Heteroduplex mobility assay (HMA) and Sanger sequencing. Online software was used to determine the percent of mosaicism in the sequenced samples. By examining the sequences of the larvae that showed a mobility shift in the HMA method, the presence of indels in the binding region of sgRNAs was confirmed, so the zebrafish model for RP disease established. Zebrafish is an ideal animal model for the functional study of various diseases involving different genes and mutations and used for evaluating different therapeutic approaches in human diseases. This study presents the production of rpe65a gene KO zebrafish models using CRISPR/Cas9 technology. This model can be used in RP pathophysiology studies and preclinical gene therapy experiments.

RESUMEN

Pathogenic variants in the RPE65 gene cause the only known form of inherited retinal degenerations (IRDs) that are prone to gene therapy. The current study is aimed at the evaluation of the prevalence of RPE65-associated retinopathy in the Russian Federation, the characterization of known variants in the RPE65 gene, and the establishment of the specificities of the mutation spectrum in Russian patients. METHODS: The analysis was carried out on blood samples obtained from 1053 non-related IRDs patients. The analysis, which consisted of 211 genes, was carried out based on the method of massive parallel sequencing (MPS) for all probands. Variant validation, as well as biallelic status verification, were carried out using direct automated Sanger sequencing. The number of copies of RPE65 exons 1-14 was analyzed with quantitative MLPA using an MRC-Holland SALSA MLPA probemix. RESULTS: Out of 1053 non-related patients, a molecular genetic diagnosis of IRDs has been confirmed in 474 cases, including 25 (5.3%) patients with RPE65-associated retinopathy. We detected 26 variants in the RPE65 gene, nine of which have not been previously described in the literature. The most common mutations in the Russian population were c.304G>T/p.(Glu102*), c.370C>T/p.(Arg124*), and c.272G>A/p.(Arg91Gln), which comprised 41.8% of all affected chromosomes. CONCLUSIONS: The current study shows that pathogenic variants in the RPE65 gene contribute significantly to the pathogenesis of IRDs and comprise 5.3% of all patients with a confirmed molecular genetic diagnosis. This study allowed for the formation of a cohort for target therapy of the disorder; such therapy has already been carried out for some patients.

Asunto(s)

Degeneración Retiniana , Humanos , Degeneración Retiniana/genética , Mutación , Exones , Biología Molecular , Federación de Rusia

RESUMEN

Our study evaluated the morphological and functional outcomes, and the side effects, of voretigene neparvovec (VN) gene therapy for RPE65-mediated inherited retinal dystrophies (IRDs) in 12 eyes (six patients) at the Oxford Eye Hospital with a mean follow-up duration of 8.2 (range 1-12) months. All patients reported a subjective vision improvement 1 month after gene therapy. Best-corrected visual acuity (BCVA) remained stable (baseline: 1.28 (±0.71) vs. last follow-up: 1.46 (±0.60); p = 0.25). Average white Full-Field Stimulus Testing (FST) showed a trend towards improvement (baseline: -4.41 (±10.62) dB vs. last follow-up: -11.98 (±13.83) dB; p = 0.18). No changes in central retinal thickness or macular volume were observed. The side effects included mild intraocular inflammation (two eyes) and cataracts (four eyes). Retinal atrophy occurred in 10 eyes (eight mild, two severe) but did not impact FST measurements during the follow-up period. Increased intraocular pressure (IOP) was noted in three patients (six eyes); four eyes (two patients) required glaucoma surgery. The overall safety and effectiveness of VN treatment in our cohort align with previous VN clinical trials, except for the higher occurrence of retinal atrophy and increased IOP in our cohort. This suggests that raised IOP and retinal atrophy may be more common than previously reported.

Asunto(s)

Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Glaucoma , Distrofias Retinianas , Humanos , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Visión Ocular , Atrofia

RESUMEN

Purpose: This study aimed to develop a prediction model to classify RPE65-mediated inherited retinal disease (IRDs) based on protein secondary structure and to analyze phenotype-protein structure correlations of RPE65 missense variants in a Chinese cohort. Methods: Pathogenic or likely pathogenic missense variants of RPE65 were obtained from UniProt, ClinVar, and HGMD databases. The three-dimensional structure of RPE65 was retrieved from the Protein Data Bank (PDB) and modified with Pymol software. A novel prediction model was developed using LASSO regression and multivariate logistic regression to identify RPE65-associated IRDs. A total of 21 Chinese probands with RPE65 variants were collected to analyze phenotype-protein structure correlations of RPE65 missense variants. Results: The study found that both pathogenic and population missense variants were associated with structural features of RPE65. Pathogenic variants were linked to sheet, ß-sheet, strands, ß-hairpins, Fe2+ (iron center), and active site cavity, while population variants were related to helix, loop, helices, and helix-helix interactions. The novel prediction model showed accuracy and confidence in predicting the disease type of RPE65 variants (AUC = 0.7531). The study identified 25 missense variants in Chinese patients, accounting for 72.4% of total mutations. A significant correlation was observed between clinical characteristics of RPE65-associated IRDs and changes in amino acid type, specifically for missense variants of F8 (H68Y, P419S). Conclusion: The study developed a novel prediction model based on the protein structure of RPE65 and investigated phenotype-protein structure correlations of RPE65 missense variants in a Chinese cohort. The findings provide insights into the precise diagnosis of RPE65-mutated IRDs.

Asunto(s)

Amaurosis Congénita de Leber , Enfermedades de la Retina , cis-trans-Isomerasas , Humanos , Amaurosis Congénita de Leber/genética , Linaje , Retina/patología , Enfermedades de la Retina/genética , Mutación Missense , cis-trans-Isomerasas/genética , Fenotipo , Conformación Proteica

RESUMEN

BACKGROUND: Inherited retinal diseases (IRDs) refer to a heterogeneous group of rare disorders that potentially lead to blindness. Emerging therapeutic options have led to a growing interest in IRDs; however, there are insufficient systematic studies on IRDs in Germany characterizing the demographics and management in clinical practice. OBJECTIVE: To characterize the care for IRD patients in Germany, to assess the applied diagnostics, the use of databases and the implementation of education in ophthalmic genetics. METHODS: The anonymous online survey (SoSci Survey GmbH) was sent to all German ophthalmology departments listed on the website of the German Ophthalmological Society and to three practices focusing on IRDs. RESULTS: The overall response rate was 44.8%. Almost all institutions (93.6%) reported seeing IRD patients, but university and non-university hospitals differed in the number of patients. Databases are used in 60% of universities but only in 5.9% of non-university hospitals. Regarding the number of patients with genetic diagnostics, 53% of the non-university and 12% of the university sites reported that 20% at most of their patients had received a molecular genetic diagnosis. The results of the IRD practices are comparable with the university hospitals. Patients with biallelic RPE65 mutations-associated IRD, potential candidates for treatment with voretigene neparvovec (Luxturna®), were followed in 9/25 participating university departments. CONCLUSION: This survey highlights the deficits in the management of IRD patients. In particular, we found a clear difference between university and non-university hospitals in the rate of patients with known molecular genetic results. Improvements should be initiated in the latter, especially because of existing and emerging therapeutic options.

Asunto(s)

Terapia Genética , Enfermedades de la Retina , Humanos , Universidades , Terapia Genética/métodos , Enfermedades de la Retina/diagnóstico , Visión Ocular , Alemania/epidemiología